Abstract: Cell motility is arguably the oldest problem in cell biology as the movement of cells was one of the first things noted when the microscope was developed in the 17th century. The last sixty years of work revealed that the molecular underpinnings of motility involve the active control of the cytoskeleton. However, many questions remain unanswered. While we have identified many of the components of the cytoskeleton and know a great deal about their biochemical and structural characteristics, we lack a systematic understanding of how the parts interact to produce coordinated cytoskeletal function such as during cell migration. Perhaps the most important problem in cell motility is understanding how cells perceive various cues in their environment and convert this information into a directed migration response. A deeper understanding of these two inter-related problems will inform higher order biological processes such as embryogenesis, immune response and wound healing, as well as diseases such as cancer metastasis. In 2012, our published a watershed paper for our research program that has shaped the course of our work ever since (Wu et al, Cell). In this paper, we discovered that mammalian fibroblasts could be nearly completely depleted of Arp2/3 by RNAi without significantly compromising viability. This allowed us to go the heart of the problem and study the function of the Arp2/3 complex directly. Using these cells and microfluidic devices to produce long-lasting gradients, we made the surprising observation that cells did not need the Arp2/3 complex for chemotaxis, but absolutely required it for haptotaxis. Inspired by our success with RNAi, we shifted to a conditional knockout mouse model where the gene encoding the critical Arpc2 (p34) subunit of Arp2/3 could be deleted on command. Using these tools, we have pursued two goals: 1) Investigate how cells can build an actin cytoskeleton without the Arp2/3 complex (funded by RO1 GM111557) and 2) Interrogate the molecular mechanisms of directed cell migration of mesenchymal cell and other cell types (funded by RO1 GM110155). Building on our progress in the last five years, we propose to extend our work in these two areas by defining the roles of Arp2/3 and non-Arp2/3 actin networks in contributing to cell behaviors such as directed migration towards chemical (chemotaxis), substrate- bound (haptotaxis) and mechanical (durotaxis) cues.